The long term of objectives of the proposed studies are to analyze DNA alterations induced by the carcinogenic aromatic amines (N-2-acetylaminofluorene and N-Methyl-4-aminoazobenzene). These studies will be aimed at determining the reaction sites, the extent of modifications, and the conformational changes in the duplex DNA. Most of these studies will be conducted on isolated nuclei rather than intact cells so that the extent of modification can be sufficiently amplified for subsequent analysis. Carcinogen-modified positions in the DNA sequence will be identified by a method to be developed. Trace components of the modified DNA will be determined by a 32P-labeling technique. DNA regions distorted as a result of the binding of the carcinogens will be defined precisely by partial digestion with single-strand specific nucleases and analyzed on high resolution sequencing gels. Thus, these highly sensitive techniques will enable to us to analyze the DNA alterations, at the sequence level, induced by the two structurally different but metabolically similar carcinogenic aromatic amines. It appears now well documented that damage to DNA plays a causal role in carcinogenesis. The proposed work will be aimed at analyzing, at the molecular level, the actions of carcinogenic aromatic amines on specific DNA sequences.